Abstract
Background
The EML4-ALK fusion gene is more frequently found in younger, never smoking, lung cancer patients. Meanwhile, never smokers exposed to secondhand tobacco smoke (SHS) during childhood are diagnosed at a younger age compared with never smoking lung cancer patients that are not exposed. We therefore hypothesized that SHS, which can induce DNA damage, is associated with the EML4-ALK fusion gene.
Methods
We compared the frequency of the EML4-ALK fusion gene among 197 never smoker lung cancer patients with and without a history of exposure to SHS during childhood at Mayo Clinic.
Results
The EML4-ALK fusion gene was detected in 33% of cases from never smokers with a history of SHS exposure during childhood, while 47% of never smoking lung cancer cases without a history of childhood SHS exposure tested positive for the fusion gene.
Conclusions
The EML4-ALK fusion gene is not enriched in tumors from individuals exposed to SHS during childhood.
Impact
These data suggest that childhood exposure to SHS is not a significant etiologic cause of the EML4-ALK fusion gene in lung cancer.
Introduction
Although they were initially thought to be common only to hematological malignancies, fusion genes caused by chromosomal rearrangements are a now recognized as a feature of solid malignancies. The anaplastic lymphoma kinase (ALK)/echinoderm microtubule-associated protein-like 4 (EML4) fusion gene was recently identified in non-small cell lung cancer (1). It is caused by an inversion of chromosome 2 and is more frequently detected in younger, never smoking patients (2). EML4-ALK-positive lung cancer represent ~5% of non-small cell lung cancer and is very sensitive to ALK inhibitors such as crizotinib (3). We (4), and others (5), previously reported that childhood exposure to secondhand tobacco smoke (SHS) is associated with a higher risk of lung cancer among adults, and that never smoker patients exposed to SHS during childhood, like patients with EML4-ALK fusion genes, are 7-15 years younger at the time of their lung cancer diagnosis compared to patients without a history of childhood SHS exposure (4). Since SHS can induce DNA damage (6) we tested the hypothesis that exposure to SHS during childhood was a potential cause of EML4-ALK formation in lung cancer.
Materials and Methods
Study Design, Sampling, and Data Collection
The 300 patient samples included in this study were selected from the Mayo Clinic Lung Cancer Cohort, an observational follow-up study (4, 7). All patients were never-smokers, defined as having smoked zero to 99 cigarettes during their lifetime. Secondhand smoke exposure data were collected via questionnaire and available for 197/300 participants. Specific information was also gathered on whether exposure was from a parent, spouse, or coworker; the number of cigarettes per day they were exposed to; and the number of years they were exposed.
EML4-ALK positivity was determined previously using two widely used and accepted assays (8). Firstly, FISH for the ALK locus rearrangement was conducted using an interphase molecular cytogenetic assay and commercially available ALK probe (Vysis, Des Plaines, IL) (8). Samples were considered positive if 15% or more of at least 100 cells counted showed splitting of the florescent probes flanking the ALK locus. ALK1 protein expression, commonly considered as a surrogate for EML4-ALK positivity, was detected by IHC using an ALK1 monoclonal antibody (Dako, Carpinteria, CA), as described previously (7). An IHC score was assigned to each case using the following criteria: Tumor section contained ≥ 10% tumor cells; score of 3 intense, granular cytoplasmic staining; score of 2, moderate, smooth cytoplasmic staining; score of 1, faint cytoplasmic staining; and score of 0, no staining (7).
For this study, EML4-ALK positivity was defined as a positive FISH score and IHC score of 2 or 3. EML4-ALK-negative samples were defined as FISH negative and an IHC score of 0 or 1. All FISH and ALK IHC interpretation was performed without the knowledge of IHC results for ALK and vice versa.
Results
Thirty-four of the 300 (11.1%) patients included in this study were positive for EML4-ALK, which was consistent with previous reports of never smokers. Among this cohort, data on exposure to SHS during childhood was available for 197 of participants. We compared the frequency of childhood SHS exposure between those positive (33%) and negative (47.1%) for EML4-ALK using a chi-square test, but did not find evidence to reject the null hypothesis, i.e., we did find an enrichment of EML4-ALK positive tumors among those with a history of childhood exposure to SHS (P=0.183) (Table 1). We further tested whether the duration of exposure, dose or source of exposure (mother versus father) was associated with EML4-ALK status, but did not find any statistically significant relationships (Table 1). Exposure to secondhand smoke during adulthood was not associated with the presence of the EML4-ALK fusion gene (Table 1).
Table 1.
Characteristics of ALK-positive and ALK-negative childhood exposure to secondhand tobacco smoke
| Positive (N=34) | Negative (N=266) | Total (N=300) | P value | |
|---|---|---|---|---|
| Any childhood ETS | 27 | 170 | 197 | 0.1831 |
| 1=YES | 9 (33.3%) | 80 (47.1%) | 89 (45.2%) | |
| 0=NO | 18 (66.7%) | 90 (52.9%) | 108 (54.8%) | |
| Years of childhood ETS | 0.6233 | |||
| N | 9 | 66 | 75 | |
| Mean (SD) | 19.8 (7.8) | 25.1 (18.0) | 24.5 (17.2) | |
| Median | 19.0 | 19.0 | 19.0 | |
| Q1, Q3 | 16.0, 19.0 | 17.0, 28.0 | 17.0, 28.0 | |
| Est pack-years child ETS | 0.4359 | |||
| N | 9 | 62 | 71 | |
| Mean (SD) | 15.3 (10.6) | 23.5 (22.2) | 22.5 (21.2) | |
| Median | 12.0 | 16.8 | 16.5 | |
| Q1, Q3 | 9.0, 28.5 | 9.0, 27.0 | 9.0, 27.0 | |
| When exposed to cig ETS | 27 | 175 | 202 | 0.0242 |
| Unknown | 0 (0.0%) | 5 (0.0%) | 5 (0.0%) | |
| 0=No ETS | 12 (44.4%) | 31 (18.2%) | 43 (21.8%) | |
| 1=Child ETS | 3 (11.1%) | 25 (14.7%) | 28 (14.2%) | |
| 2=Adult ETS | 6 (22.2%) | 59 (34.7%) | 65 (33.0%) | |
| 3=Life ETS | 6 (22.2%) | 55 (32.4%) | 61 (31.0%) | |
| Any father ETS | 27 | 175 | 202 | 0.3500 |
| 1=YES | 9 (33.3%) | 75 (42.9%) | 84 (41.6%) | |
| 0=NO | 18 (66.7%) | 100 (57.1%) | 118 (58.4%) | |
| Amount of father ETS | 27 | 175 | 202 | 0.4634 |
| 0=NONE | 18 (66.7%) | 104 (59.4%) | 122 (60.4%) | |
| 1=LIGHT | 3 (11.1%) | 20 (11.4%) | 23 (11.4%) | |
| 2,3=MODERATE~HEAVY | 6 (22.2%) | 51 (29.1%) | 57 (28.2%) | |
| Any mother ETS | 27 | 175 | 202 | 0.3390 |
| 1=YES | 1 (3.7%) | 21 (12.0%) | 22 (10.9%) | |
| 0=NO | 26 (96.3%) | 154 (88.0%) | 180 (89.1%) | |
| Amount of mother ETS | 27 | 175 | 202 | 0.2852 |
| 0=NONE | 26 (96.3%) | 156 (89.1%) | 182 (90.1%) | |
| 1=LIGHT | 1 (3.7%) | 2 (1.1%) | 3 (1.5%) | |
| 2,3=MODERATE~HEAVY | 0 (0.0%) | 17 (9.7%) | 17 (8.5%) |
Discussion
In recent years the discovery of fusion genes in cancer has accelerated, in part due to the advent of next generation sequencing technologies. These discoveries have improved outcomes for many patients given that many are potent driver oncogenes and sensitive to targeted therapies. However the etiologic cause of such fusion genes, for the most part, remains unknown. In this study, we hypothesized that the EML4-ALK fusion gene is caused by exposure to SHS during childhood. We based this hypothesis on the observation that; a) lung cancer patients with the EML4-ALK fusion gene and those exposed to SHS during childhood are both diagnosed with lung cancer at a younger age, and b) exposure to SHS can cause DNA damage. We did not find statistical evidence that the EML4-ALK is associated with a history of SHS exposure during childhood. Potential limitations of this study include the possibility of recall bias among those reporting childhood SHS exposure. While it is possible that other fusion genes could be caused by childhood exposure to SHS, our data do not support the hypothesis that the EML4-ALK fusion gene is associated with SHS exposure during childhood.
Acknowledgments
This work was supported by the Intramural Program of the Centre for Cancer Research, National Cancer Institute, NIH grants NIH-R01-CA80127 (P. Yang) NIH-R01-CA84354 (P. Yang), and NIH-R01-CA115857 (P. Yang). J. Jen is a recipient of the New Investigator Award from the American Cancer Society and supported by funding from Mayo Clinic Cancer Center and the Center for Individualized Medicine. P. Yang, J. Jen, E.S. Yi and Y. Wang received support from The Mayo Clinic Foundation.
Footnotes
Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.
References
- 1.Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561–6. doi: 10.1038/nature05945. [DOI] [PubMed] [Google Scholar]
- 2.Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clinical cancer research : an official journal of the American Association for Cancer Research. 2009;15:5216–23. doi: 10.1158/1078-0432.CCR-09-0802. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pao W, Girard N. New driver mutations in non-small-cell lung cancer. The lancet oncology. 2011;12:175–80. doi: 10.1016/S1470-2045(10)70087-5. [DOI] [PubMed] [Google Scholar]
- 4.Olivo-Marston SE, Yang P, Mechanic LE, Bowman ED, Pine SR, Loffredo CA, et al. Childhood exposure to secondhand smoke and functional mannose binding lectin polymorphisms are associated with increased lung cancer risk. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2009;18:3375–83. doi: 10.1158/1055-9965.EPI-09-0986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.The Health Consequences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon General. Atlanta (GA): 2006. [PubMed] [Google Scholar]
- 6.Husgafvel-Pursiainen K. Genotoxicity of environmental tobacco smoke: a review. Mutation research. 2004;567:427–45. doi: 10.1016/j.mrrev.2004.06.004. [DOI] [PubMed] [Google Scholar]
- 7.Yang P, Kulig K, Boland JM, Erickson-Johnson MR, Oliveira AM, Wampfler J, et al. Worse disease-free survival in never-smokers with ALK+ lung adenocarcinoma. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2012;7:90–7. doi: 10.1097/JTO.0b013e31823c5c32. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Yi ES, Boland JM, Maleszewski JJ, Roden AC, Oliveira AM, Aubry MC, et al. Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: IHC score algorithm for FISH. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2011;6:459–65. doi: 10.1097/JTO.0b013e318209edb9. [DOI] [PubMed] [Google Scholar]